biostudies-arrayexpressJernej UleHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15648SLAM-seq (thiol(SH)-linked alkylation for the metabolic sequencing of RNA) was applied to human primary CD8⁺ T cells subjected to in vitro activation with αCD3/αCD28 beads. To quantify mRNA half-lives and their dynamic changes during CD8⁺ T cell activation, we implemented a pulse-chase design with 24 hours of labelling using 100 µM 4-thiouridine (4SU), replenished every 4 hours with fresh RPMI medium containing 100 µM 4SU. This was followed by uridine washout for 0, 0.5, 1, 3, 6, and 16 hours. Samples were collected from non-activated, Day1-activated, and Day5-activated CD8⁺ T cells at each washout step (2 donors per time point). These datasets provide transcriptome-wide measurements of mRNA half-lives and reveal how mRNA turnover alters upon T cell activation.biostudies-arrayexpressLibrary Construction - SLAM-Seq libraries were prepared following established protocols (Herzog et al., Nat Methods 2017), with minor modifications. Briefly, thiol-modified RNA was alkylated to introduce T>C mutations detectable in sequencing. Libraries were prepared from extracted total RNA using the Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit FWD, V2 (Cat. No. 191.96, Lexogen GmbH), which generates forward-stranded Illumina-compatible libraries of sequences close to the 3’ end of polyadenylated RNA. The protocol involves oligo(dT)-primed reverse transcription from the 3′ poly(A) tail of mRNAs, followed by RNA degradation and synthesis of the second strand using random primers. The second strand contains Illumina-compatible linker sequences, ensuring strand specificity. After PCR amplification and purification, the resulting cDNA libraries were quantified and quality-checked by Bioanalyzer before sequencing.Sequencing - Libraries were sequenced on an Illumina HiSeq 4000 by the Advanced Sequencing Facility at The Francis Crick Institute.Nucleic Acid Extraction - Total RNA was extracted from human CD8⁺ T cells after each labelling and chase time point using TRIzol reagent, followed by column-based purification. RNA integrity was assessed by Bioanalyzer (Agilent).Sample Treatment - Human CD8+ cells were either non-activated or activated with αCD3/αCD28 beads (1:1 bead-to-cell ratio, Gibco 11132D). During cell culture, CD8+ cells were labelled with 100 µM 4-thiouridine (4SU; Sigma T4509), replenished every 4 h for 24 h. After labelling, CD8+ cells were washed and chased with 100 mM uridine (Sigma U3750) for 0, 0.5, 1, 3, 6, or 16 hours. Day 1 and day 5 activated CD8⁺ T cells were processed in parallel, with beads removed at day 4 for the latter. At each chase time point, 1-2 × 10⁶ cells were collected for RNA extraction.Growth Protocol - Human CD8⁺ T cells were cultured in RPMI-1640 medium (Gibco, 52400-025) supplemented with 10% FBS, 1% penicillin–streptomycin, and 30 U/ml IL-2 (Roche). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂.Sample Collection - Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors through Cambridge Bioscience, NHS Blood and Transplant (UK). Ethical approval and written informed consent were obtained in accordance with the Declaration of Helsinki (2013). PBMCs were isolated using Ficoll-Paque PLUS density gradient centrifugation. Human CD8⁺ T cells were purified from PBMCs using MACS Miltenyi kits (total CD8⁺ T cells: 130-096-495; MicroBeads: 130-045-201; FcR blocking reagent: 130-059-901) according to the manufacturer’s instructions.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Data analysis was performed using the SLAMdunk pipeline (v0.3.3) within Snakemake (v5.3). Reads were demultiplexed with Cutadapt (v1.18), quality filtered (Q20), barcode trimmed, and mapped to the human genome (GRCh38) with Slamdunk map using local alignment. Reads were filtered for ≥95% identity and ≥50% mapping. SNPs were called with VarScan2 (v2.4.1), and T>C conversions overlapping SNPs were removed. Conversions were counted in sliding windows, normalized by T content and coverage, and assessed with Slamdunk Alyoop. Transcript half-lives were estimated by fitting T>C decay curves to a monoexponential model using the R package minpack.lm (v1.2-86), yielding robust half-life estimates (R² > 0.5) across conditions.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 4000RNA-seq of coding RNAHomo sapiensJernej UlePaulo GameirofalseSLAM-seq analysis of mRNA half-lives in human CD8⁺ T cells during activationSLAM-seq (thiol(SH)-linked alkylation for the metabolic sequencing of RNA) was applied to human primary CD8⁺ T cells subjected to in vitro activation with αCD3/αCD28 beads. To quantify mRNA half-lives and their dynamic changes during CD8⁺ T cell activation, we implemented a pulse-chase design with 24 hours of labelling using 100 µM 4-thiouridine (4SU), replenished every 4 hours with fresh RPMI medium containing 100 µM 4SU. This was followed by uridine washout for 0, 0.5, 1, 3, 6, and 16 hours. Samples were collected from non-activated, Day1-activated, and Day5-activated CD8⁺ T cells at each washout step (2 donors per time point). These datasets provide transcriptome-wide measurements of mRNA half-lives and reveal how mRNA turnover alters upon T cell activation.2025-10-15T00:00:00Z2026-05-27T15:48:26.77Z2025-10-01T10:30:52.531ZE-MTAB-15648ERP180861E-MTAB-15643EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969